This project focuses on the role that cytoskeletal forces play in platelet adhesions to mediate the formation and stability of clots. Platelet adhesion significantly influences thrombosis since platelets need to aggregate together to stop blood loss, but also to detach and disassemble in order to not obstruct flow to other tissue. It is unclear how platelets direct this dual response, but previous work in this area has shown that integrin adhesions and cytoskeletal forces both are necessary for controlling thrombus stability. However, the interactions between integrins and forces are not completely understood, in part because there are limited tools and techniques available to simultaneously monitor the mechanical interactions that occur at the subcellular level. This project will combine our expertise in measuring cellular forces and quantitative image analysis in order to study the relationship between cytoskeleton-generated platelet forces and integrin adhesivity in platelets. The approach involves the use of flexible post force-sensors with nanoscale dimensions that can directly measure cytoskeletal forces at focal adhesions of platelets and immunofluorescence image analysis to assess their cytoskeletal protein organization. The long-term goal of this project will be to develop a new method that quantitatively measures the magnitude of platelet forces without the confounding influences of platelet-fibrin interactions. As a proof-of-concept, this approach will be used to identify and characterize cytoskeletal proteins that promote platelet adhesion and thrombus stability. Specific Aim 1 will be to measure platelet forces using flexible nanopost force-sensors. Specific Aim 2 will be to investigate the development of platelet forces of activated platelets. Specific Aim 3 will be to examine the relationship of platelet forces with focal adhesion development and lay the groundwork for mechanistic studies of force and integrin interactions that regulate platelet adhesion. The device to be developed will enable physiological studies of platelets at the nanoscale that have not been possible previously. The increased understanding that this new approach will bring will have the potential to provide important input into efforts to control thrombosis, which is a critical event in the development of heart disease and stroke. PUBLIC HEALTH RELEVANCE: The device to be developed will enable physiological studies of platelets at the nanoscale that have not been possible previously. The increased understanding that this new approach will bring will have the potential to provide important input into efforts to control thrombosis, which is a critical event in the development of heart disease and stroke.